Objective To investigate the application value of cellular antomatic DNA image cytometry （DNA-ICM） in cervical cancer screening. Methods Ten cases of normal cervical tissues with diploid DNA content were selected as the control group; 69 cases of no intraepithelial or malignant lesions （NILM）, 30 cases of low grade squamous intraepithelial leisons （LSIL）, 65 cases of high grade squamous intraepithelial leisions （HSIL）, and 28 cases of squamous cell carcinoma （SCC） were selected as the experimental groups. Pap staining cytological screening, automatic DNA-ICM and ploidy analysis after Feulgen staining, and Ki-67 immunocytochemical staining were performed. Results The sensitivity of DNA-ICM and HPV SIL+ screening was 65.9% and 84.6% （χ²=34.217, P<0.001）, and the specificity was 72.5% and 55.1% （χ²=10.340, P = 0.006） respectively. The DI thresholds of NILM, LSIL, HSIL, and SCC were ≤4, ≤4, ≤6, and ≤5, respectively. The average number of anploid cells was 0.9, 3.9, 9.4, and 12.0, and the average number of Ki-67 positive cells was 3.1, 10.5, 88.6, and 255.6, respectively, with a significant difference between the two groups （χ²=14.680, P<0.05）. The proportion of few （≤2） anploid cells was 10.1%, 33.3%, 26.1%, and 28.6%, respectively. The detection rates of cell DNA heteroploidy in the low, medium, and high risk HPV groups were 12.1%, 57.5%, and 82.3%, respectively, with a significant difference among the groups （χ²=43.420, P<0.001）. Conclusion DNA index （DI） reflects the structural and quantitative changes of DNA during the cell cycle. The number and DI threshold of DNA anploid cells have important application values for cytological diagnosis, evaluation of proliferation activity and progression of lesions.